Serum Concentration of Thyroid Hormones Long-Term after Sulfur Mustard Exposure.

Background
Despite several reports on the clinical manifestations of sulfur mustard (SM) intoxication, there is no study on serum concentrations of thyroid hormones long-term after SM exposure. In this study, the changes in thyroid functioning parameters 20 yr after SM exposure were evaluated.


Methods
This study is a part of a larger historical cohort study conducted in 2007 following 20 years of the exposure to SM, called Sardasht-Iran cohort study (SICS). We (SICS) comprised an SM-exposed group from Sardasht City, West Azerbaijan Province, Iran (n=169 as hospitalized group and n=203 as non-hospitalized exposed group); and control participants were selected from Rabat, a town near Sardasht (n=126). Peripheral blood samples were taken in fasting state and then the sera were separated. T4, T3, TSH, antithyroglobulin (anti-Tg), and antithyroid peroxidase (anti-TPO) concentrations in the sera were measured by the ELISA method.


Results
The mean of T3 concentration was significantly higher in the exposed than control group (0.88 ± 0.26 nmol/L vs 0.8 ± 0.25 nmol/L, P<0.001). The levels of TSH, T4, and T3up were not significantly different between the exposed and control groups. Thyroglobulin level was significantly higher in the exposed non-hospitalized group (56.07 ± 140.22 μg/L vs 17.66 ± 41.49 μg/L, P=0.004), but the level of anti-Tg and anti-TPO showed no significant differences between the two groups.


Conclusion
More studies are needed on the alterations in thyroid hormones, their gene expressions, and mechanisms involved in SM exposure to clarify the causes of these alterations.


Introduction
Sulfur mustard [bis(2-chloroethyl) sulfide; SM], also known as mustard gas, was initially used as a vesicant chemical warfare agent during World War I. According to the report of the specialists appointed by the Secretary-General of the United Nations, mustard gas was extensively used by Iraq during the conflict against Iran (1). Sardasht, a border town in the northwest of Iran, was exposed to sulfur mustard in 1987 by the Iraqi army. In addition to acute extensive multi-organ problems, there are long-term adverse health effects of exposure to mustard gas (2)(3)(4). Despite a fair number of reports on clinical manifestations of SM intoxication, there are few studies on paraclinical and molecular parameters and systemic effects of SM long term after exposure. Therefore, the underlying molecular mechanisms are less known. Various complaints and clinical presentations of the survived people seeking medical care and review of the literature support such concerns, even though no systematic scientific evidence has been available (5). A decline in mean serum-free T4 index (FT4I) and free T3 index was reported less than one month after exposure (6). Moreover, they explained the time course of serum concentration of thyroid hormones. Free T4 and T3 indices decreased and reverse T3 (rT3) increased in the first week following exposure. Except for an increase in FT4I and a decrease in thyroid-stimulating hormone (TSH) by the third week, serum hormone concentrations remained unchanged until the fifth week after injury. They concluded that exposure to chemical weapons containing SM results in alterations in serum concentrations of thyroid hormones. There is no study on serum concentrations of thyroid hormones long-term after SM exposure. In this study, the changes of thyroid function parameters 20 years after SM exposure were evaluated.

Study Design and Participants
This study is a part of a larger historical cohort study conducted in 2007 following 20 years of the exposure to SM, called Sardasht, West Azerbaijan Province, Iran cohort study (SICS), of which the details of the study design and methods have been previously reported (7). Briefly, the exposed group were male individuals from Sardasht categorized into two major subgroups based on severity of injuries, just after exposure. SM-exposed patients hospitalized based on the severity of injuries after acute exposure were considered as the hospitalized group (n=169) who had a history of at least one-week admission in hospital, and patients with mild and subclinical problems not hospitalized after acute exposure as the non-hospitalized exposed group (n=203). Control participants were selected from Rabat, a town near Sardasht, concurrently with the exposed group. Moreover, the control samples included men matched with the study group by age (n=126). The age range covered by the study was 20-60 yr. Both study groups were compared in terms of marital status, level of education, employment, and smoking status.

Ethical approval
The study was approved by the Ethical Committee of the Board of Research Ethics of Janbazan Medical and Engineering Research Center (JMERC), the Board of Research of the Ministry of Health and Medical Education, and Shahed University. Volunteers who signed an informed consent were recruited.

Serum Preparation
Peripheral blood samples were taken in the morning in fasting state using Vacationer tubes (BD Biosciences), 20 years following SM exposure in the exposed group and, alongside, in the control group. After clotting, the sera were separated by a 20 min centrifugation at 2000×g in 4 °C, aliquoted and stored at −80 °C until test.

Statistical Analysis
Statistical analysis was done using the SPSS (Windows, ver. 16.0, Chicago, IL, USA). Values are given as mean (± SD). Comparison of thyroid factors between study groups was done with ANOVA (Tukey's post hoc). Comparison of the normal range was done with chi-square test. A Pvalue of less than 0.05 was considered statistically significant.

Results
The mean age of the exposed group was 44 ± 11 yr. The result revealed insignificant differences in terms of age, marital status, and smoking status in contrast to significant differences in terms of level of education and, and employment, which were higher among the exposed group (Table 1).  The mean of T3 concentration was significantly higher in the exposed group (0.88±0.26 nmol/L) compared to the control (0.80 ± 0.25) (P<0.001); however, these values were still within normal clinical range. The levels of TSH, T4, T3 up, anti-Tg and anti-TPO were not significantly differ-ent between the exposed and control groups. Regarding two subgroups of exposure (hospitalized and non-hospitalized), the patterns of measured parameters were similar. Thyroglobulin level was higher in the exposed but not hospitalized group (56.07±140.22 µg/L, P=0.004), compared to the control group. The free T3 index, calculated from the serum total of T3 and T3 uptake, was significantly elevated in the exposed group similar to the T3 level. The results of this study showed that the ratio of T3 to T4 in the exposed group was significantly higher than the control group (P= 0.006) ( Table 4).

Discussion
The aim of this study was to determine the circulating levels of thyroid hormones 20 years after SM exposure. The mean concentrations of T4 and TSH in the exposed group were not significantly different from the control group. Whereas serum T3 concentrations and T3/T4 ratio exhib-ited significant increase in SM-exposed people compared to the control group; however, it was within normal clinical range. Severity of injury had no effect on this hormone level. We also assessed TPO, Tg and anti-Tg. The data showed elevated levels of Tg in the SM-not hospitalized exposed group compared to the control group. However, the level of anti-Tg showed no significant differences between the two groups. Anti-TPO was similar in both groups. To our knowledge, this is the first report pointing to circulating levels of thyroid hormones, 20 years after SM exposure. So far there has been no study on the long-term effects of SM exposure on these parameters to compare. Serum concentrations of thyroid hormones were measured in the first, third, and fifth week following injury in the SM-exposed group compared to the control group, in order to evaluate the time course of changes in serum concentrations of thyroid hormones, cortisol, and adrenocorticotropic hormone (ACTH) in patients exposed to chemical weapons containing SM (6). In the first week after exposure, free T4 and T3 indices decreased and rT3, cortisol, and ACTH increased. Thereafter, serum hormone concentrations remained unchanged until the fifth week after injury, except for an increase in FT4I, a decrease in TSH by the third week, and a steady decline in serum cortisol. In the current study 20 years following exposure to SM, we found significantly higher levels of T3 in the exposed group compared to the control group, whereas, T4 and TSH levels were not statistically significant. This result is in contrast to acute effects of SM exposure, demonstrated in the foregoing study. The time interval of the post-exposure could be reasonable justification for this dissimilar hormone level. However, other undiscovered pathophysiological mechanisms also should be considered.
Since thyroid function tests in the SM-exposed people in the other studies were assessed short term after exposure, there was no comparable data. Anyway, consistent with the short-term findings (6), no changes were seen in the serum levels of T4 and TSH in our study, 20 years after SM exposure.
The delayed toxic effects of SM were documented in Iranian veterans, focusing on head and neck complications. For the first time, they reported carcinomas of thyroid and nasopharynx in patients with SM exposure (8). Despite the decrease in T3 level in most of these reports, different elucidations could interpret the elevation of T3 in our study; one is that more subjects in our control group have lower level of T3 than normal range (11.8% in control and 4.3% in exposed). On the other hand, there are numerous human and animal studies on the variety of chemicals that disrupt thyroid hormone homeostasis. Such differences in T3 and T3/T4 ratio were observed in exposure to contaminant chemicals such as PCBs in fish (9). In vertebrates, the main hormone produced by thyroid, prohormone T4, is converted to T3 in peripheral target tissue cells by 5-deiodinase (10). The differences in T3 and T3/T4 ratio may point to alterations in the peripheral conversion of T4 into T3. Some studies on fish suggested that pesticides and other chemicals may alter 5deiodinase activity, which can lead to increased T3 and decreased T4 (4-6, 11-13). Although there has been no study on the cellular and molecular mechanisms involved in SM-induced thyroid function disorders, there are a large number of studies on endocrine-disrupting chemicals (EDCs). Numerous mechanisms have been proposed, including changes in the expression of a large number of potential target genes or proteins, altered by EDCs (14). Activation of EDCs may interfere with thyroid function by interfering with thyroid hormone synthetic pathways, deiodinase functions in peripheral tissues, altering TH turnover, and carrying proteins in the blood (6,8,13,(15)(16)(17)(18)(19). However, the limitation of this study is that as our subjects were selected from a previous large cohort study, detailed clinical evaluation in view of specific thyroid assessment was not performed. Further insight into the role of SM in the pathogenesis of thyroid requires deep researches on the cellular and molecular mechanisms involved. This would enable both researchers and clinicians to provide more accurate evaluation and therapeutic strategies when dealing with sufferers of SM exposure. More studies are needed on the alterations in thyroid hormones, their gene expressions, and involved mechanisms in SM exposure to clarify the causes of these alterations.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.